Antimicrobial peptides are crucial for host defense at mucosal surfaces. Bacterial factors responsible for induction of human ␤-defensin-2 (hBD-2) mRNA expression in Caco-2 human carcinoma cells were determined. Salmonella enteritidis, Salmonella typhimurium, Salmonella typhi, Salmonella dublin, and culture supernatants of these strains induced hBD-2 mRNA expression in Caco-2 human carcinoma cells. Using luciferase as a reporter gene for a ϳ2.1-kilobase pair hBD-2 promoter, the hBD-2-inducing factor in culture supernatant of S. enteritidis was isolated. The supernatant factor was heat-stable and proteinase-sensitive. After purification by anion exchange and gel filtration chromatography, the hBD-2-inducing factor was identified as a 53-kDa monomeric protein with the amino-terminal sequence AQVINTNSLSLLTQNNLNK, which is identical to that of the flagella filament structural protein (FliC) of S. enteritidis. Consistent with this finding, the 53-kDa protein reacted with anti-FliC antibody, which prevented its induction of hBD-2 mRNA in Caco-2 cells. In agreement, the hBD-2-inducing activity in culture supernatant was completely neutralized by anti-FliC antibody. In gel retardation analyses, FliC increased binding of NF-B (p65 homodimer) to hBD-2 gene promoter sequences. We conclude that S. enteritidis FliC induces hBD-2 expression in Caco-2 cells via NF-B activation and thus plays an important role in up-regulation of the innate immune response.Antimicrobial peptides play an important role in host defense at mucosal surfaces. The two major groups of vertebrate defensins, ␣-and ␤-defensins, differ in the arrangements of their disulfide bonds. Six human ␣-defensins (HD-1 to HD-6) 1 and two ␤-defensins (hBD-1 and hBD-2) have been reported to date. HD-1, HD-2, HD-3, and HD-4 are present in neutrophils, where they constitute 30 -50% of the total protein in azurophilic granules (1). HD-5 and HD-6 were identified in the Paneth cells of small intestinal crypts (2, 3) and in female reproductive tissue (4). hBD-1 was purified from plasma (5) and detected in a range of epithelial tissues (6). hBD-2 was purified from skin and shown to be expressed in the lung and uterus (7). hBD-2 was induced in mucosal tissues following bacterial infections (7-9).Salmonella species are Gram-negative organisms that cause gastroenteritis and enteric fever in humans. As pointed out by Bä umler et al. (10), an increase in numbers of human infections with Salmonella enteritidis began in the 1960s but was followed by an almost 50% decrease from 1970 to 1976. Another increase began in 1977, with signs of a decrease beginning in 1992. In contrast, the increasing frequency of S. enteritidis infection in poultry did not begin until 1989, and a decline started in 1996 (11). Reasons for the fluctuations are unknown, but the recent recognition of food-borne infection with S. enteritidis has renewed interest in how these organisms can invade, persist, and spread.To evaluate the role of defensins in S. enteritidis infection, we investigated the induction of ...

Vibrio cholerae is an aetiological agent of cholera that inhabits marine and estuarine environments. It can survive harsh environments by entering the viable but non-culturable (VBNC) state, but the related changes in gene expression have not been described. Here, we experimentally induced the VBNC state in V. cholerae O1, by incubation in artificial seawater at 4°C. Bacterial cells that were incubated for 70 days retained their membrane integrity and were pathogenic, colonizing the gut of iron-dextran-treated mice, even though they formed no colonies on tryptic soy agar (TSA) or TSA amended with pyruvate. We therefore used this stage of cells as the VBNC bacteria. We compared the global transcription pattern of the VBNC cells with that of stationary-phase cells grown in rich medium. A total of 100 genes were induced by more than fivefold in the VBNC state, and the modulated genes were mostly those responsible for cellular processes. Furthermore, real-time RT-PCR analysis verified the changes in the expression levels, showing that the VC0230 [iron(III) ABC transporter], VC1212 (polB), VC2132 (fliG) and VC2187 (flaC) mRNAs were increased in the non-culturable state. Thus, these genes may be suitable markers for the detection of VBNC V. cholerae. To our knowledge, this is the first report of a comprehensive transcriptome analysis of V. cholerae in the VBNC state. The significance of this gene expression profile compared with those of in vivo isolates and non-stressed bacteria (culturable in vitro) is its potential to provide information about the public health risk from dormant bacteria.

Campylobacter jejuni is a microaerophilic bacterium that causes diarrhea in humans. The first step in establishing an infection is adherence to a host cell, which involves two major cell-binding proteins, Peb1A (CBF1) and Peb4 (CBF2). Because the functional role of Peb4 on the cell adhesion remains unclear compared with that of Peb1A, a C. jejuni peb4 deletion mutant was constructed and cell adherence and ability to colonize mouse intestine were studied. The result showed that adherence of the peb4 mutant strain to INT407 cells was 1-2% that of the wild-type strain. Mouse challenge experiments showed a reduced level and duration of intestinal colonization by the mutant compared with the wild-type strain. In addition, fewer peb4 mutant cells than wild-type cells responded to stress by forming a biofilm. Proteomic analysis revealed that the expression levels of proteins involved in various adhesion, transport, and motility functions, which are required for biofilm formation by the pathogen, were lower in the peb4 mutant than in the wild-type strain. A Peb4 homolog has prolyl cis/trans-isomerase activity, suggesting that the loss of this activity in the mutant strain may be responsible for the repression of these proteins.

SummaryMulticellular biofilms are an ancient bacterial adaptation that offers a protective environment for survival in hostile habitats. In microaerophilic organisms such as C ampylobacter, biofilms play a key role in transmission to humans as the bacteria are exposed to atmospheric oxygen concentrations when leaving the reservoir host gut. Genetic determinants of biofilm formation differ between species, but little is known about how strains of the same species achieve the biofilm phenotype with different genetic backgrounds. Our approach combines genome‐wide association studies with traditional microbiology techniques to investigate the genetic basis of biofilm formation in 102 C ampylobacter jejuni isolates. We quantified biofilm formation among the isolates and identified hotspots of genetic variation in homologous sequences that correspond to variation in biofilm phenotypes. Thirteen genes demonstrated a statistically robust association including those involved in adhesion, motility, glycosylation, capsule production and oxidative stress. The genes associated with biofilm formation were different in the host generalist ST‐21 and ST‐45 clonal complexes, which are frequently isolated from multiple host species and clinical samples. This suggests the evolution of enhanced biofilm from different genetic backgrounds and a possible role in colonization of multiple hosts and transmission to humans.

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains isolated from wild deer in Japan were examined. A total of 43 fecal samples were collected 4 times from 4 different sites around Obihiro City, Hokkaido, Japan, in June and July 1997. Seven STEC strains were isolated by PCR screening, all of them were confirmed by ELISA and Vero cell cytotoxicity assay to be producing only active Stx type 2 (Stx2). Moreover, they seemed to carry the hemolysin and eaeA genes of STEC 0157:H7, and some isolates harbored large plasmids which were similar to the 90-kilobase virulence plasmid of STEC 0157:H7. Based on their plasmid profiles, antibiotic resistance patterns, PCR-based DNA fingerprinting data obtained by using random amplified polymorphic DNA (RAPD) and the stx2 gene sequences, all isolates were divergent from each other except for 3 isolates from the first and second samplings. A DNA sequence analysis of representative isolates revealed that deer originating STEC strains were closely related to each other, but not to the Stx2-producing STEC strains isolated from a mass outbreak in Obihiro at the same time. A phylogenic analysis of the deduced Stx2 amino acid sequences demonstrated that three distinct clusters existed in the deer originating STEC strains and that the Stx of deer originating STEC was closely associated with that originating from humans, but not those of STEC originating from other animals. These results suggest that STEC contamination of deer carcasses should be considered as a potential source of human infection and adequate sanitary inspection of meat for human consumption is also essential for wild animals.

Summary Helicobacter pyloriis a bacterial pathogen that colonizes the gastric niche of ~50% of the human population worldwide and is known to cause peptic ulceration and gastric cancer. Pathology of infection strongly depends on a cag pathogenicity island (cagPAI)-encoded type IV secretion system (T4SS). Here, we aimed to identify as yet unknown bacterial factors involved in cagPAI effector function and performed a large-scale screen of an H. pylori transposon mutant library using activation of the pro-inflammatory transcription factor NF-κB in human gastric epithelial cells as a measure of T4SS function. Analysis of ~3000 H. pylori mutants revealed three non-cagPAI genes that affected NF-κB nuclear translocation. Of these, the outer membrane protein HopQ from H. pylori strain P12 was essential for CagA translocation and for CagA-mediated host cell responses such as formation of the hummingbird phenotype and cell scattering. Besides that, deletion of hopQ reduced T4SS-dependent activation of NF-κB, induction of MAPK signalling and secretion of interleukin 8 (IL-8) in the host cells, but did not affect motility or the quantity of bacteria attached to host cells. Hence, we identified HopQ as a non-cagPAI-encoded co-factor of T4SS function.

An outbreak caused by salted salmon roe contaminated with enterohemorrhagic Escherichia coli O157 occurred in Japan in 1998. Since about 0.75 to 1.5 viable cells were estimated to cause infection, we presumed that O157 might enter the viable but nonculturable (VNC) state in salted salmon roe and consequently that viable cell numbers might be underestimated. Although patient-originating O157 cells could not grow on agar plates after 72 h of incubation in 13% NaCl, they were resuscitated in yeast extract broth, and more than 90% of the cells were shown to be viable by fluorescent staining, suggesting that almost all of them could enter the VNC state in NaCl water. Roe-originating O157 was resistant to NaCl because it could grow on agar after 72 h of incubation in NaCl water, but about 20% of cells appeared to enter the VNC state. Therefore, germfree mice were infected with O157 to examine the resuscitation of cells in the VNC state and the retention of pathogenicity. O157 that originated in roe, but not patients, killed mice and was isolated from the intestine. However, these isolates had become sensitive to NaCl. O157 cells of roe origin incubated in normal media also killed mice and were isolated from the intestine, but they also became transiently NaCl sensitive. We therefore propose that bacterial cells might enter the VNC state under conditions of stress, such as those encountered in vivo or in high salt concentrations, and then revive when those conditions have eased. If so, the VNC state in food is potentially dangerous from a public health viewpoint and may have to be considered at the time of food inspection. Finally, the establishment of a simple recovery system for VNC cells should be established.In Japan, salmon roe is soaked in a liquid seasoning, which consists of soy sauce (79.0%), water (14.0%), chemical seasoning (6.5%), synthetic sake (0.3%), and a fermented seasoning (0.2%); its salt content is equal to a 13% NaCl concentration. This salted salmon roe is a popular component of Japanese sushi. The preservation of foods by high salt concentrations has been viewed historically as an effective means of preventing food-borne infections (11, 13). However, an outbreak caused by salted salmon roe which was contaminated with enterohemorrhagic Escherichia coli (EHEC) O157 occurred in four independent places in Japan in 1998, with 62 cases reported (1). Since all the causative foods were manufactured by the same company, the salmon roe was probably contaminated with O157 during the production process. Although the definitive source of O157 could not be identified because the roe was stored frozen for 9 months, it appeared that O157 could survive freezing and a high concentration of NaCl (22) and retain its pathogenicity for humans. In addition, it was proved by the most probable number method that about 0.75 to 1.5 viable cells of O157 could cause infection (1). This number was considered to be very low for infection (23). Recently, it has been reported that Vibrio cholerae, E. coli, Campylobacter jejuni, ...

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 was isolated for the first time from Musca domestica L. A total of 310 fly samples was collected from 4 different farms in Obihiro-City, Hokkaido, in the summer and autumn of 1997;5 samples carried E. coli serotype O157:H7. Using ELISA and Vero cell cytotoxicity assay, 3 isolates from 1 cattle farm produced both active Shiga-toxin type 1 (Stx1) and 2 (Stx2). These isolates also carried hemolysin and eaeA genes and harbored the 90-kb virulence plasmid of EHEC O157:H7. Based on plasmid profiles, antibiotic patterns, polymerase chain reaction (PCR)-based DNA finger printing analysis using random amplified polymorphic DNA, pulsed field gel electrophoresis analysis, and DNA sequences of stx1 and stx2, all 3 isolates from fly samples were identical. These results indicate that the house fly is capable of carrying the toxigenic EHEC O157:H7 involved in human disease.